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Rational Targeting of Cellular Cholesterol in Diffuse Large BCell Lymphoma (DLBCL) Enabled by Functional Lipoprotein Nanoparticles: A Therapeutic Strategy Dependent on Cell of Origin Jonathan S. Rink, Shuo Yang, Osman Cen, Tim Taxter, Kaylin M. McMahon, Sol Misener, Amir Behdad, Richard Longnecker, Leo I Gordon, and C. Shad Thaxton Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00710 • Publication Date (Web): 21 Sep 2017 Downloaded from http://pubs.acs.org on September 22, 2017

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Molecular Pharmaceutics

Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled by Functional Lipoprotein Nanoparticles: A Therapeutic Strategy Dependent on Cell of Origin Jonathan S. Rink1,2, Shuo Yang3, Osman Cen3, Tim Taxter4,5, Kaylin M. McMahon1,2,5, Sol Misener1, Amir Behdad4, Richard Longnecker6, Leo I. Gordon3,7* and C. Shad Thaxton1,2,7,8* 1: Department of Urology, Northwestern University, Chicago, IL, USA 60611 2: Simpson Querrey Institute for BioNanotechnology, Northwestern University, Chicago, IL, USA 60611 3: Division of Hematology/Oncology, Department of Medicine, Northwestern University, Chicago, IL, USA 60611 4: Department of Pathology, Northwestern University, Chicago, IL, USA 60611 5: Developmental Therapeutic Institute, Northwestern University, Chicago, IL, USA 60611 6: Department of Microbiology and Immunology, Northwestern University, Chicago, IL USA, 60611 7: Robert H Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL, USA 60611

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8: International Institute for Nanotechnology (IIN), Northwestern University, Evanston, IL, USA, 60208 KEYWORDS: Lymphoma, Cholesterol, High-density lipoproteins, Nanoparticles, B-cell receptor signaling

ABSTRACT

Cancer cells have altered metabolism and, in some cases, an increased demand for cholesterol. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative approach. Toward this end, we focused on diffuse large B-cell lymphoma (DLBCL) as a model because there is differential cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol uptake through the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). As we previously reported, GC DLBCL are exquisitely sensitive to HDL NP as monotherapy while ABC DLBCL are less sensitive. Herein, we report that enhanced BCR signaling and resultant de novo cholesterol synthesis in ABC DLBCL drastically reduces the ability of HDL NPs to reduce cellular cholesterol and induce cell death. Therefore, we combined HDL NP with the BCR signaling inhibitor ibrutinib and the SYK inhibitor R406. By targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis, we achieved cellular cholesterol reduction and induced apoptosis in otherwise resistant ABC DLBCL cell lines. These results in lymphoma demonstrate that


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reduction of cellular cholesterol is a powerful mechanism to induce apoptosis. Cells rich in cholesterol require HDL NP therapy to reduce uptake and molecularly targeted agents that inhibit upstream pathways that stimulate de novo cholesterol synthesis, thus, providing a new paradigm for rationally targeting cholesterol metabolism as therapy for cancer.

Introduction Normal, healthy human cells tightly regulate cellular cholesterol in order to maintain cell membrane integrity and to stabilize membrane-anchored signaling pathways.1,

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Evidence

suggests a major role for lipids in the progression of hematological3-5 and other malignancies6-10 with specific involvement of cholesterol and cholesteryl esters, along with the high-density lipoproteins (HDL) that carry them.11-14 In the case of B-cell lymphoma, B-cell receptor (BCR) signaling, including low level, tonic signaling observed in germinal center (GC)-derived diffuse large B cell lymphoma (DLBCL) and chronic active signaling in activated B-cell (ABC) DLBCL, has been shown to directly promote cholesterol biosynthesis through intermediate kinases downstream of the BCR to maintain cell membrane integrity and BCR signaling.15 Perturbation of BCR-induced de novo cholesterol biosynthesis in malignant B cells, for example by inhibiting or depleting SYK (spleen associated tyrosine kinase), disrupts the cell membrane and inhibits BCR signaling, leading to apoptosis.15 Further delineation of this mechanism, and identification of targeted therapy that enables specific disruption of cholesterol metabolism in malignant B cells and other cholesterol-dependent cancer cells, may provide important new insights and more rational cancer treatment.16-19 The high-affinity HDL receptor, scavenger receptor type B1 (SCARB1)20 is a potential target for perturbing cellular cholesterol homeostasis. SCARB1 mediates bidirectional


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cholesterol flux between HDL particles and the cell membrane and provides a conduit for delivery of cholesteryl ester from the core of natural HDLs to target cells. In addition to mediating cholesterol flux, binding of natural HDL to SCARB1 activates second messenger signaling pathways, including the PI3K-AKT pro-survival pathway in multiple cell types, including breast cancer and endothelial cells.21-23 Our group previously reported that a large number of cancer cell lines, including B cell lymphoma, express SCARB1, while peripheral naïve and memory B-cells do not.19 Accordingly, SCARB1 expression in malignant cells may provide a critical opportunity to specifically and therapeutically reduce cellular cholesterol. Synthetic, bio-inspired HDL-like nanoparticles (HDL NPs) represent a first-in-class therapy that specifically bind SCARB1 and can perturb cellular cholesterol and cholesteryl ester homeostasis, ultimately depleting cellular cholesterol and cholesteryl esters when compared with natural, cholesterol-rich HDLs.16, 18, 24, 25 HDL NPs consist of a 5nm diameter gold nanoparticle core surface-functionalized with the HDL-defining apolipoprotein A1 (apoA-I) and a phospholipid bilayer.26, 27 HDL NPs are similar to natural, mature, spherical HDL with regard to surface composition, negative surface charge, size, and shape.16,

24, 25

Functionally, HDL NPs

mediate the efflux of free cholesterol from cells through SCARB1.16, 19 and, owing to the core gold nanoparticle, are not an appreciable source of cholesteryl ester.18, 19 These unique features enable HDL NPs to target SCARB1, but result in cellular cholesterol reduction when compared with natural HDLs.19 Importantly, HDL NPs induce profound cell death in GC DLBCL cell lines but are relatively ineffective against ABC DLBCL cells.19 However, a better understanding of the mechanism of action of HDL NPs and how this therapy can be rationally combined with other agents remains unexplored.1, 22, 28-30


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Given the differences in BCR signaling levels and sensitivity to HDL NP, we hypothesized that ABC DLBCL cells are resistant to HDL NP-induced cholesterol depletion due to enhanced BCR-associated de novo cholesterol synthesis. Effective therapy may be realized by combining HDL NPs with agents that target BCR signaling. Our data confirm that targeted cellular cholesterol depletion is necessary to induce apoptosis in lymphoma cells, which provides a rational approach to target cholesterol metabolism in other cancer types that are cholesterol dependent. Experimental Methods Cell lines and authentication: The human cell lines Jurkat and SUDHL4 were obtained from ATCC and were used within 3 months of receipt and/or resuscitation. ATCC uses the Short Tandem Repeat (STR) profiling method to authenticate cell lines. The human cell lines TMD8 and HBL-1 were generously provided by Dr. Louis Staudt, whose laboratory uses the novel DNA copy number variance fingerprinting technique to verify authenticity.31 Additionally, the STR profiles for TMD8 and HBL-1 cells were analyzed by ATCC and did not match any profile in their database. Cell culture: SUDHL4 and Jurkat cells were cultured in RPMI 1640 with L-glutamine and 25mM HEPES (Mediatech) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals), and 1% PenStrep, at 37 °C with 5% CO2 in a humidified incubator, except TMD8 and HBL-1, which were cultured with 15% FBS. The SCARB1 blocking and rabbit IgG isotype control antibodies were obtained from Novus Biologicals. The PI3K inhibitor Pilaralisib (XL147; Selleck Chem), the AKT inhibitor GDC-0068 (Selleck Chem), the SYK inhibitor R406 (Selleck Chem) and the BTK inhibitor ibrutinib (PCI-32756; Selleck Chem) were added to cells


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at the same time as HDL NP treatment. The cholesterol sequesterant, methyl-β-cyclodextran (MβCD; Sigma Aldrich), was added to cells at the same time as HDL NPs, with concentrations ranging from 156.25 µM to 10 mM. Western blot analysis: Western blot analysis was conducted as previously described.19 The SCARB1 (ab52629) and SREBP-1a (ab3259) antibodies was obtained from Abcam, and PARP (#9542) and β-actin (#4970) antibodies were obtained from Cell Signaling Technologies. HDL NP synthesis and quantification: HDL NPs were synthesized and quantified as previously described.16, 18, 24, 32 Briefly, 5 nm diameter gold nanoparticles (AuNPs) are surface functionalized with apolipoprotein A-I (added at a 5 fold excess relative to [AuNP]) for one hour,

followed

by

addition

of

the

phospholipids

1,2-dipalmitoyl-sn-glycero-3-

phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (PDP PE) and 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) overnight (each added at 250 fold excess to the [Au NP]). The HDL NPs were purified using the KrosFlo TFF (Tangential Flow Filtration) system, with a 50 kDa cut-off PES column. To generate fluorescently labeled HDL NPs (DiI HDL NP), 1,1’Dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine percholorate (DiI; ThermoFisher) was incorporated into the HDL NP synthesis. DiI was added at a 1µM final concentration (12.5-fold molar excess to AuNP) between addition of the PDP PE and DPPC phospholipids. Purification of DiI HDL NPs was conducted as described above for non-fluorescently HDL NPs, with special care being taken to protect the HDL NPs from light during synthesis and purification. UV/Vis spectroscopy was used to measure the concentration of the HDL NPs (ε = 9.696 x 106 M-1cm-1 for 5 nm diameter AuNPs). The size and surface charge of HDL NPs and DiI HDL NPs were determined using dynamic light scattering (DLS) and zeta potential, respectively, using a Malvern Zetasizer.


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MTS assay: MTS assays were carried out as previously described19, and according to manufacturer’s instructions (CellTiter assay, Promega). Lymphoma cells were re-suspended at a concentration of 2 x 105 cells/ml, and 80 µl of cells added to each well of a 96 well plate. Cells were then treated with HDL NPs and/or various inhibitors (20 µl) for 72 hours prior to addition of the MTS reagent. DiI HDL NP cell binding/ uptake assay: Lymphoma cells were plated at a concentration of 2.5 x 105 cells/ml and treated with 5-25 nM of fluorescently-labeled DiI HDL NPs for 24 hours. Cells were then washed twice with 1 X PBS, re-suspended in cold FACS buffer (PBS, 1% bovine serum albumin, 0.1% sodium azide) and cell associated fluorescence was obtained using the BD LSR II Fortessa flow cytometer. Data were analyzed using the FCS Express software. Cholesterol efflux assays: Lymphoma cell cholesterol efflux assays were conducted as previously described.16, 19, 24 Lymphoma cells were labeled with 1,2 3H-cholesterol (1µCi/mL) for 24 hours in standard culture media. Post labeling, cells were spun down, re-suspended in serum free RPMI 1640, and then plated into 24 well culture plates at a concentration of 1.5 x 105 cells/well. HDL NPs and human HDL were added at a final concentration of 50 nM, with or without the addition of the SCARB1 blocking antibody (1:250), or the rabbit IgG isotype control antibody (1:250). Cells were incubated at 37oC for 4 hours. The cells were spun down and the supernatants collected, filtered and analyzed by liquid scintillation counting (countssupernatant). Total cholesterol labeling was quantified by extracting the radiolabeled cholesterol using isopropanol from lymphoma cells at the start of HDL NP and human HDL treatment (countstotal). The background cholesterol efflux in the absence of any cholesterol acceptor (e.g. hHDL or HDL NP) was subtracted from each value and the percent cholesterol efflux calculated by dividing the countssupernatant by the countstotal and multiplying by 100.


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Total cholesterol quantification: Total cholesterol was quantified using the Amplex Red Cholesterol Assay (Thermo Fisher Scientific) according to manufacturer’s instructions. Briefly, lysates were prepared from SUDHL4, TMD8 and HBL-1 cells treated with PBS, HDL NPs, Ibrutinib, or HDL NP + Ibrutinib for 24 hours. Cholesterol content values were standardized to total cell number, as measured using the Countess automated cell counter (Invitrogen). Phos-flow analysis of p-AKT and p-BTK. Cells were cultured with or without HDL NPs (50nM, 100nM), Ibrutinib (10nM) or PBS for 2 hours, then washed with FBS Stain Buffer (BD Biosciences). The cells were fixed and permeabilized with BD Phosflow Lyse/Fix buffer and BD Perm Buffer III according to manufacturer’s instructions. Following permeabilization, the cells were washed twice with Stain Buffer and then incubated for 60 minutes at room temperature with AlexaFluor 647-labeled p-AKT or p-BTK antibodies. The cells were washed twice and resuspended in Stain Buffer for flow cytometric analysis on the BD LSR II Fortessa flow cytometer. Data were analyzed using FCS Express software. RT-qPCR analysis: SUDHL4, TMD8 and HBL-1 cells were treated with HDL NPs (10nM, 50nM), hHDL (50nM) or PBS for 48 hours, and RNA isolated using the RNeasy Mini kit (Qiagen). RNA samples (500ng RNA per 30µl reaction) were reverse transcribed using a TaqMan Reverse Transcription kit, and qPCR for ACAT2, DHCR7, INSIG1, HMGCS1, LSS, MSMO1, MVD, SCD, p21, and SQLE run on a BioRad iCycler. Samples were standardized to β-actin, and relative expression was calculated using the ∆∆Ct method. For every condition, biological triplicates were run. Mouse tumor xenograft models: All animal work was conducted in accordance with Northwestern University’s IACUC and CCM facilities under an approved animal protocol. Flank


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tumors were initiated using the SUDHL4 and TMD8 cell line in SCID-beige mice (Taconic) and allowed to progress until reaching a size of approximately 50mm3. For the SUDHL4 study, mice were randomly divided into 3 treatment groups, based on their initial tumor volume: PBS, HDL NP 3 times per week or HDL NPs 5 times per week. HDL NPs (100µl of 1µM HDL NP) were injected I.V. for 21 days, at which time the mice were euthanized and blood was collected for blood chemistry analysis. For the TMD8 tumor xenograft study, mice were randomly divided into 4 treatment groups based on tumor volume at time of treatment initiation: PBS, HDL NP (1µM), Ibrutinib (3mg/kg body weight), or HDL NP (1µM) + Ibrutinib (3mg/kg body weight) and received 100µl of treatment 5 times per week for two weeks. Mice were euthanized on the day after the last dose of treatment. Statistics: One-way ANOVAs and 2-sided student’s t-test were used to determine statistical significance, and the data were graphed using the GraphPad Prism software. Synergy calculations were performed using the Calcusyn (Biosoft) software. Results HDL NPs Induce Lymphoma Cell Death. We tested the sensitivity of two commonly used ABC DLBCL cell lines, TMD8 and HBL-1, to HDL NP relative to the sensitive GC DLBCL SUDHL4 cell line. Both the GC and ABC DLBCL cell lines were positive for SCARB1 expression (Figure S1). All B-cell lymphoma cell lines showed a dose-dependent loss of viability when treated with HDL NPs, but not when treated with human HDL (Figure 1a). The ABC DLBCL cell lines were significantly less sensitive to the HDL NP as compared with the GC DLBCL line SUDHL4 (Figure 1a). The SCARB1-negative Jurkat cell line was not sensitive to HDL NP (Figure 1a). This sensitivity correlated to HDL NP-induced PARP cleavage, with


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increased signal observed in the SUDHL4 cells compared with TMD8 and HBL-1 cells (Figure S2). Because of differences in cell killing with HDL NPs, we tested if there was differential binding/ uptake of the HDL NPs in GC vs ABC DLBCL cells. To this end, we incubated SUDHL4 and TMD8 cells with HDL NPs labeled with the fluorescent intercalating dye DiI (DiI HDL NPs) for 24 hours prior to flow cytometric analysis. DiI HDL NPs were similar to HDL NPs in terms of their size (12.1 ± 0.3 nm diameter for HDL NP vs. 12.9 ± 0.7 nm for DiI HDL NP), surface charge (-45.9 ± 3.2 mV vs. -50.0 ± 0.2), stability and ability to induce GC DLBCL cell death (Figure S3a, b). Interestingly, the TMD8 cells displayed enhanced DiI HDL NP fluorescence compared to SUDHL4 cells (Figure 3c-e). These data demonstrate that the mechanism by which the HDL NPs differentially induce apoptosis in GC vs ABC DLBCL cells cannot be explained by increased HDL NP binding/ uptake. HDL NPs Modulate Cellular Cholesterol Through SCARB1. Our previous work demonstrated that HDL NPs target and modulate cholesterol flux through SCARB1.16, 19, 24, 25 Therefore, we investigated the ability of HDL NPs to efflux cholesterol from GC and ABC DLBCL cells, with particular attention paid to SCARB1-mediated efflux. Both SUDHL4 and TMD8 cells effluxed cholesterol after exposure to HDL NPs (Figure 1b, c). Inhibition of HDL NP binding to SCARB1 by addition of a blocking antibody partially reduced cholesterol efflux to HDL NPs, which was not seen when an isotype control antibody was added (Figure 1b, c), suggesting that the nanoparticles engage with SCARB1 and modulate cholesterol flux through the receptor.


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Figure 1: HDL NPs induce B cell lymphoma cell death through modulation of cellular cholesterol. (a) HDL NPs (solid line) potently induce cell death in the GC DLBCL cell line SUDHL4, but not the ABC DLBCL cell lines TMD8 or HBL-1. Human HDL (dotted line) does not induce lymphoma cell death. *p

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